Whole genome and transcriptome integrated analyses guide clinical care of pediatric poor prognosis cancers.

The role for routine whole genome and transcriptome analysis (WGTA) for poor prognosis pediatric cancers remains undetermined. Here, we characterize somatic mutations, structural rearrangements, copy number variants, gene expression, immuno-profiles and germline cancer predisposition variants in children and adolescents with relapsed, refractory or poor prognosis malignancies who underwent somatic WGTA and matched germline sequencing. Seventy-nine participants with a median age at enrollment of 8.8 y (range 6 months to 21.2 y) are included. Germline pathogenic/likely pathogenic variants are identified in 12% of participants, of which 60% were not known prior. Therapeutically actionable variants are identified by targeted gene report and whole genome in 32% and 62% of participants, respectively, and increase to 96% after integrating transcriptome analyses. Thirty-two molecularly informed therapies are pursued in 28 participants with 54% achieving a clinical benefit rate; objective response or stable disease ≥6 months. Integrated WGTA identifies therapeutically actionable variants in almost all tumors and are directly translatable to clinical care of children with poor prognosis cancers.

Development and evaluation of time-resolved rapid immunofluorescence test for detection of TSOL18 specific antibody in porcine cysticercosis infections.

BACKGROUND: Porcine cysticercosis, a serious zoonotic parasitic disease, is caused by the larvae of Taenia solium and has been acknowledged by the World Organization for Animal Health. The current detection methods of Cysticercus cellulosae cannot meet the needs of large-scale and rapid detection in the field. We hypothesized that the immunofluorescence chromatography test strip (ICS) for detecting Cysticercus cellulosae, according to optimization of a series of reaction systems was conducted, and sensitivity, specificity, and stability testing, and was finally compared with ELISA. This method utilizes Eu3+-labeled time-resolved fluorescent microspheres (TRFM) coupled with TSOL18 antigen to detect TSOL18 antibodies in infected pig sera. RESULTS: ICS and autopsy have highly consistent diagnostic results (n = 133), as determined by Cohen's κ analysis (κ = 0.925). And the results showed that the proposed ICS are high sensitivity (0.9459) with specificity (0.9792). The ICS was unable to detect positive samples of other parasites. It can be stored for at least six months at 4℃. CONCLUSIONS: In summary, we established a TRFM-ICS method with higher sensitivity and specificity than indirect ELISA. Results obtained from serum samples can be read within 10 min, indicating a rapid, user-friendly test suitable for large-scale field detection.

Discovery of Novel Isoquinoline Analogues as Dual Tubulin Polymerization/V-ATPase Inhibitors with Immunogenic Cell Death Induction.

Cancer immunotherapy has revolutionized clinical advances in a variety of cancers. Due to the low immunogenicity of the tumor, only a few patients can benefit from it. Specific microtubule inhibitors can effectively induce immunogenic cell death and improve immunogenicity of the tumor. A series of isoquinoline derivatives based on the natural products podophyllotoxin and diphyllin were designed and synthesized. Among them, F10 showed robust antiproliferation activity against four human cancer cell lines, and it was verified that F10 exerted antiproliferative activity by inhibiting tubulin and V-ATPase. Further studies indicated that F10 is able to induce immunogenic cell death in addition to apoptosis. Meanwhile, F10 inhibited tumor growth in an RM-1 homograft model with enhanced T lymphocyte infiltration. These results suggest that F10 may be a promising lead compound for the development of a new generation of microtubule drugs.

Engine gas path component fault diagnosis based on a sparse deep stacking network.

Accurate engine gas path component fault diagnosis methods are key to ensuring the reliability and safety of engine operations. At present, the effectiveness of the data-driven gas path component fault diagnosis methods has been widely verified in engineering applications. The deep stack neural network (DSN), as a common deep learning neural network, has been gaining more attention in gas path fault diagnosis studies. However, various gas path component faults with strong coupling effects could occur simultaneously, resulting the DSN method less effective for engine gas path fault diagnosis. In order to improve the prediction performance of the DSN handling multiple gas path component fault diagnosis, a sparse regularization and representation method was proposed. The sparse regularization term is used to expand the traditional deep stacking neural network in the sparse representation, and the predicted output tag is close to the target output tag through this term. The diagnosis performance of six different neural network methods were compared by various engine gas path component fault diagnosis types. The results show that the proposed sparse regularization method significantly improves the prediction performance of the DSN, with an accuracy rate 99.9% under various gas path component fault conditions, which is higher than other methods. The proposed engine gas path component fault diagnosis method can handle multiple coupling gas path faults, and help engine operators to develop maintenance plans for the purpose of engine health management.

Discovery of Novel Potent Covalent Glutathione Peroxidase 4 Inhibitors as Highly Selective Ferroptosis Inducers for the Treatment of Triple-Negative Breast Cancer.

Glutathione peroxidase 4 (GPX4) is a promising target to induce ferroptosis for the treatment of triple-negative breast cancer (TNBC). We designed and synthesized a novel series of covalent GPX4 inhibitors based on RSL3 and ML162 by structural integration and simplification strategies. Among them, compound C18 revealed a remarkable inhibitory activity against TNBC cells and significantly inhibited the activity of GPX4 compared to RSL3 and ML162. Moreover, it was identified that C18 could notably induce ferroptosis with high selectivity by increasing the accumulation of lipid peroxides (LPOs) in cells. Further study demonstrated that C18 covalently bound to the Sec46 of GPX4. Surprisingly, C18 exhibited an outstanding potency of tumor growth inhibition in the MDA-MB-231 xenograft model with a TGI value of 81.0%@20 mg/kg without obvious toxicity. Overall, C18 could be a promising GPX4 covalent inhibitor to induce ferroptosis for the treatment of TNBC.

Extracorporeal membrane oxygenation for thyroid surgery in patients with severe tracheal stenosis.

Venovenous extracorporeal membrane oxygenation is effective for maintaining gas exchange in patients with respiratory failure or severe tracheal stenosis. Perioperative anesthetic management of severe airway obstruction can be associated with ventilation or intubation difficulties. Consequently, venovenous extracorporeal membrane oxygenation could be an option for treating such patients to avoid potential risks. However, only a limited number of similar cases have been reported. Therefore, we have summarized two cases to provide theoretical and practical references for treating patients with respiratory failure or severe tracheal stenosis using extracorporeal membrane oxygenation.

Discovery of Novel N-Heterocyclic-Fused Deoxypodophyllotoxin Analogues as Tubulin Polymerization Inhibitors Targeting the Colchicine-Binding Site for Cancer Treatment.

Natural products are a major source of anticancer agents and play critical roles in anticancer drug development. Inspired by the complexity-to-diversity strategy, novel deoxypodophyllotoxin (DPT) analogues were designed and synthesized. Among them, compound C3 exhibited the potent antiproliferative activity against four human cancer cell lines with IC50 values in the low nanomolar range. Additionally, it showed marked activity against paclitaxel-resistant MCF-7 cells and A549 cells. Moreover, compound C3 can inhibit tubulin polymerization by targeting the colchicine-binding site of tubulin. Further study revealed that compound C3 could arrest cancer cells in the G2/M phase and disrupt the angiogenesis in human umbilical vein endothelial cells. Meanwhile, C3 remarkably inhibited cancer cell motility and migration, as well as considerably inhibited tumor growth in MCF-7 and MCF-7/TxR xenograft model without obvious toxicity. Collectively, these results indicated that compound C3 may be a promising tubulin polymerization inhibitor development for cancer treatment.

Sloping Farmlands Conversion to Mixed Forest Improves Soil Carbon Pool on the Loess Plateau.

Vegetation restoration is considered a potentially useful strategy for controlling soil erosion and improving soil organic carbon (SOC) in arid and semiarid ecosystems. However, there is still debate regarding which vegetation restoration type is the best choice. In this study, four vegetation restoration types (i.e., grasslands, shrubs, forests and mixed forests) converted from sloping farmlands were selected to explore the SOC variation among the four types and to investigate which soil factors had the greatest effect on SOC. The results showed while the magnitude of effect differed between vegetation restoration type, all studied systems significantly increased SOC and labile organic carbon contents (p < 0.01), soil nutrients such as total nitrogen (TN) (p < 0.01), available nitrogen (AN) (p < 0.01), total phosphorus (TP) (p < 0.05) and available phosphorus (AP) (p < 0.05), soil enzyme activities such as phosphatase (p < 0.01), soil microbial biomass carbon (MBC) (p < 0.05), and basal respiration (BR) (p < 0.05), but had significant negative correlationswith polyphenol oxidase (p < 0.05). However, the effects of vegetation restoration of farmland converted to natural grasslands, shrubs, forests and mixed forests varied. Among the types studied, the mixed forests had the largest overall positive effects on SOC overall, followed by the natural grasslands. Soil nutrients such as N and P and soil microbial activities were the main factors that affected SOC after vegetation restoration. Mixed forests such as Robinia pseudoacacia and Caragana korshinskii are the best choice for farmland conversion on the central of the Loess Plateau.

Design, synthesis, and biological evaluation of novel diphenylamine derivatives as tubulin polymerization inhibitors targeting the colchicine binding site.

A novel series of diphenylamine derivatives were designed and synthesized, and their biological activities were evaluated. The anti-proliferative activities of the derivatives were tested against five human cancer cell lines (MCF-7, MDA-MB-231, A549, HeLa and HT29). Among them, compound 5f exhibited the promising anti-proliferative activity against HT29 cell lines with the IC50 value of 23 nM. Further biological studies depicted that compound 5f inhibited cancer cell migration, colony formation and angiogenesis. Besides, dynamics studies and molecular docking studies revealed that compound 5f inhibited tubulin polymerization which may be a result of the compound binding to the colchicine site of tubulin. Furthermore, compound 5f arrested HT29 cell cycle at G2/M phase, and induced HT29 cell apoptosis by upregulating cyclin B1, Bcl-2, Bax, Cleaved-caspase9, Cleaved-caspase3, PARP, Cleaved-PARP proteins, and downregulating p-cdc25c (S216), p-cdc2 (T15) proteins. Mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) were also determined to confirm the cell apoptosis process. Finally, compound 5f greatly inhibited the tumor growth in HT29 xenograft mice by 75.5% at 10 mg/kg. Meanwhile, compound 5f owned the good pharmacokinetic properties. All the results promised that 5f is of potential to act as an antitumor candidate and worthy of further investigation.

A Scalable Strand-Specific Protocol Enabling Full-Length Total RNA Sequencing From Single Cells.

RNA sequencing (RNAseq) has been widely used to generate bulk gene expression measurements collected from pools of cells. Only relatively recently have single-cell RNAseq (scRNAseq) methods provided opportunities for gene expression analyses at the single-cell level, allowing researchers to study heterogeneous mixtures of cells at unprecedented resolution. Tumors tend to be composed of heterogeneous cellular mixtures and are frequently the subjects of such analyses. Extensive method developments have led to several protocols for scRNAseq but, owing to the small amounts of RNA in single cells, technical constraints have required compromises. For example, the majority of scRNAseq methods are limited to sequencing only the 3' or 5' termini of transcripts. Other protocols that facilitate full-length transcript profiling tend to capture only polyadenylated mRNAs and are generally limited to processing only 96 cells at a time. Here, we address these limitations and present a novel protocol that allows for the high-throughput sequencing of full-length, total RNA at single-cell resolution. We demonstrate that our method produced strand-specific sequencing data for both polyadenylated and non-polyadenylated transcripts, enabled the profiling of transcript regions beyond only transcript termini, and yielded data rich enough to allow identification of cell types from heterogeneous biological samples.

Radiomics Signature: A potential biomarker for the prediction of survival in Advanced Hepatocellular Carcinoma.

Objectives: To develop and validate radiomics nomograms for the pretreatment predictions of overall survival (OS) and time to progression (TTP) in the patients with advanced hepatocellular carcinoma (HCC) treated with apatinib plus transarterial chemoembolization (TACE), and to assess the incremental value of the clinical-radiomics nomograms for estimating individual OS and TTP. Methods: A total of 60 patients with advanced HCC (BCLC stage C) treated with apatinib plus TACE were divided into a training set (n=48) and a validation set (n=12). The predictors identified from the clinical variables and the radiomics signature constructed from the computed tomography images, such as ɑ-fetoprotein level (AFP), formfactor, the grey level co-occurrence matrix, the gray level size zone matrix, and the gray level run-length matrix, were used to build the clinical-radiomics nomograms and the radiomics nomograms for the prediction of OS and TTP. Results: Apatinib plus TACE benefited the patients with advanced HCC, with a 579-day median OS and a 270-day median TTP. The nomograms were built with the radiomics signature and AFP, and achieved favorable prediction efficacy with acceptable calibration curves. Decision curve analyses demonstrated that the clinical-radiomics nomograms outperformed the radiomics nomograms for the predictions of OS and TTP. Conclusions: Apatinib plus TACE may improve OS and prolonged TTP in the patients with advanced HCC. The clinical-radiomics nomograms, a noninvasive pretreatment prediction tool that incorporate radiomics signature and AFP, demonstrated good prediction accuracy for OS and TTP in these patients. These results indicate that the clinical-radiomics nomograms may provide novel insight for precise personalized medicine approaches in the patients with advanced HCC.

Whole-slide laser microdissection for tumour enrichment.

The practical application of genome-scale technologies to precision oncology research requires flexible tissue processing strategies that can be used to differentially select both tumour and normal cell populations from formalin-fixed, paraffin-embedded tissues. As tumour sequencing scales towards clinical implementation, practical difficulties in scheduling and obtaining fresh tissue biopsies at scale, including blood samples as surrogates for matched 'normal' DNA, have focused attention on the use of formalin-preserved clinical samples collected routinely for diagnostic purposes. In practice, such samples often contain both tumour and normal cells which, if correctly partitioned, could be used to profile both tumour and normal genomes, thus identifying somatic alterations. Here we report a semi-automated method for laser microdissecting entire slide-mounted tissue sections to enrich for cells of interest with sufficient yield for whole genome and transcriptome sequencing. Using this method, we demonstrated enrichment of tumour material from mixed tumour-normal samples by up to 67%. Leveraging new methods that allow for the extraction of high-quality nucleic acids from small amounts of formalin-fixed tissues, we further showed that the method was successful in yielding sequence data of sufficient quality for use in BC Cancer's Personalized OncoGenomics (POG) program. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Uncovering Clinically Relevant Gene Fusions with Integrated Genomic and Transcriptomic Profiling of Metastatic Cancers.

PURPOSE: Gene fusions are important oncogenic drivers and many are actionable. Whole-genome and transcriptome (WGS and RNA-seq, respectively) sequencing can discover novel clinically relevant fusions. EXPERIMENTAL DESIGN: Using WGS and RNA-seq, we reviewed the prevalence of fusions in a cohort of 570 patients with cancer, and compared prevalence to that predicted with commercially available panels. Fusions were annotated using a consensus variant calling pipeline (MAVIS) and required that a contig of the breakpoint could be constructed and supported from ≥2 structural variant detection approaches. RESULTS: In 570 patients with advanced cancer, MAVIS identified 81 recurrent fusions by WGS and 111 by RNA-seq, of which 18 fusions by WGS and 19 by RNA-seq were noted in at least 3 separate patients. The most common fusions were EML4-ALK in thoracic malignancies (9/69, 13%), and CMTM8-CMTM7 in colorectal cancer (4/73, 5.5%). Combined genomic and transcriptomic analysis identified novel fusion partners for clinically relevant genes, such as NTRK2 (novel partners: SHC3, DAPK1), and NTRK3 (novel partners: POLG, PIBF1). CONCLUSIONS: Utilizing WGS/RNA-seq facilitates identification of novel fusions in clinically relevant genes, and detected a greater proportion than commercially available panels are expected to find. A significant benefit of WGS and RNA-seq is the innate ability to retrospectively identify variants that becomes clinically relevant over time, without the need for additional testing, which is not possible with panel-based approaches.

Continuous positive airway pressure effectively ameliorates arrhythmias in patients with obstructive sleep apnea-hypopnea via counteracting the inflammation.

INTRODUCTION: This work is aimed at evaluating the therapeutic effect of continuous positive airway pressure (CPAP) in treatment of patients with obstructive sleep apnea-hypopnea syndrome (OSAHS) combined with arrhythmias as well as clarifying the possible mechanism underpinning such an intervention. METHODS: Through exclusions, a total of 108 OSAHS patients combined with arrhythmias were enrolled from June 2017 to June 2019 with full clinical information in this work. A computerized permuted block design with varying block stratification and size according to age, sex, AHI and type of arrhythmia was used to randomize 108 patients to CPAP versus sham CPAP for a period of 12-week. All were subjected to unchanged pharmacological anti-arrhythmia therapy combined with CPAP. Before and after CPAP treatment, the improvement of various arrhythmias was compared between the CPAP group and the sham-CPAP group. The levels of CRP, IL-6 and TNF-ɑ were measured simultaneously. RESULTS: During follow-up, the mean (±SD) CPAP pressure used in the CPAP group was 12.3 (±3.1) cm H2O. The use of CPAP and sham CPAP was on average of 5.2 ± 0.56 and 5.1 ± 0.63 h/night, respectively. After 12 weeks of CPAP therapy, the AHI was significantly decreased and the lowest blood oxygen saturation was notably elevated in the CPAP group compared to the sham-CPAP group, P < 0.05. The CPAP therapy, compared with the sham-CPAP group, significantly reduced the incidence of all types of arrhythmia in patients with OSAHS. The level of the c-reactive protein (CRP), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) was significantly lower in the CPAP group than in the sham-CPAP group (P < 0.05). Pearson correlation analysis showed that the reduction in the incidence of total arrhythmias was positively correlated with the decrease of CRP, IL-6 and TNF-ɑ levels, respectively. CONCLUSION: Findings from this work suggest that proper use of CPAP significantly benefits to OSAHS patients combined with arrhythmias, possibly via counteracting the inflammation.

Effects of carbon nanotubes on the toxicities of copper, cadmium and zinc toward the freshwater microalgae Scenedesmus obliquus.

Due to their unique structure and properties, carbon nanotubes (CNTs) released into the aquatic environment can potentially influence the behavior of other coexisting pollutants, thereby altering their toxicity to aquatic organisms. In this study, the toxicities of multi-walled CNTs and three heavy metals, copper (Cu), cadmium (Cd) and zinc (Zn) were determined individually. Following this, CNTs with low concentrations (1 and 5 mg/L) were co-exposed with Cu, Cd or Zn to the microalgae Scenedesmus obliquus, to investigate the effects and underlying mechanisms of CNTs on metal toxicity. Results showed that CNTs, especially at a concentration of 5 mg/L, promoted algae growth and enhanced photosynthetic efficiency via increasing exciton trap efficiency and quantum yield for electron transport. Introduction of CNTs appeared to alleviate the adverse effects of Cu, Cd or Zn on microalgae, indicated by algae growth, total chlorophyll content and photosynthetic indices. However, these effects differed greatly for different metals, depending on both the toxicity of each metal and the exposure period (4 day and 8 day). Enhancement of photosynthesis and interference of metal uptake by CNTs, have a crucial role in the effects of CNTs on metal toxicity.

Author Correction: Synthesis and characterization of polyethylene glycol-phenol-formaldehyde based polyurethane composite.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Pan-cancer analysis of advanced patient tumors reveals interactions between therapy and genomic landscapes.

Advanced and metastatic tumors with complex treatment histories drive cancer mortality. Here we describe the POG570 cohort, a comprehensive whole-genome, transcriptome and clinical dataset, amenable for exploration of the impacts of therapies on genomic landscapes. Previous exposure to DNA-damaging chemotherapies and mutations affecting DNA repair genes, including POLQ and genes encoding Polζ, were associated with genome-wide, therapy-induced mutagenesis. Exposure to platinum therapies coincided with signatures SBS31 and DSB5 and, when combined with DNA synthesis inhibitors, signature SBS17b. Alterations in ESR1, EGFR, CTNNB1, FGFR1, VEGFA and DPYD were consistent with drug resistance and sensitivity. Recurrent noncoding events were found in regulatory region hotspots of genes including TERT, PLEKHS1, AP2A1 and ADGRG6. Mutation burden and immune signatures corresponded with overall survival and response to immunotherapy. Our data offer a rich resource for investigation of advanced cancers and interpretation of whole-genome and transcriptome sequencing in the context of a cancer clinic.

Synthesis and characterization of polyethylene glycol-phenol-formaldehyde based polyurethane composite.

A series of phenol-formaldehyde-polyethylene glycol polyether polyols (PF-PEGs) were synthesized through the condensation polymerization and etherification of phenol, formaldehyde, and poly(ethylene glycol) (PEG) under alkaline conditions and subsequently reacted with 1,6-hexamethylene diisocyanate to obtain polyurethane (PU) films using acetone as solvents. The influence of phenol and formaldehyde to PEG mass ratio ((P + F)/PEG) on the hydroxyl number of PF-PEGs and mechanical properties, thermal stabilities, crystallization behaviors, as well as microstructure of polyurethane composite films were studied using chemical analysis, mechanical tests, thermogravimetric analyses (TGA), dynamic mechanical analyses (DMA), X-ray diffraction (XRD), scanning and transmission electron microscopies (SEM and TEM), respectively. Results demonstrated that PF-PEGs with (P + F)/PEG of 50/50 had the highest hydroxyl number of 323 mg K(OH)/g. The incorporation of phenol and formaldehyde into PEG improved the mechanical properties of polyurethane films, glass transition temperature (Tg), and thermal properties but resulted in the brittleness characteristic of the composite films and low crystallization properties. Moreover, the synthesis mechanism of PF-PEGs polyurethane composite films was revealed, which would provide a theoretical base for the preparation of the rigid polyurethane foams based on phenolic resins.

Application of a Neural Network Whole Transcriptome-Based Pan-Cancer Method for Diagnosis of Primary and Metastatic Cancers.

Importance: A molecular diagnostic method that incorporates information about the transcriptional status of all genes across multiple tissue types can strengthen confidence in cancer diagnosis. Objective: To determine the practical use of a whole transcriptome-based pan-cancer method in diagnosing primary and metastatic cancers and resolving complex diagnoses. Design, Setting, and Participants: This cross-sectional diagnostic study assessed Supervised Cancer Origin Prediction Using Expression (SCOPE), a machine learning method using whole-transcriptome RNA sequencing data. Training was performed on publicly available primary cancer data sets, including The Cancer Genome Atlas. Testing was performed retrospectively on untreated primary cancers and treated metastases from volunteer adult patients at BC Cancer in Vancouver, British Columbia, from January 1, 2013, to March 31, 2016, and testing spanned 10 822 samples and 66 output classes representing untreated primary cancers (n = 40) and adjacent normal tissues (n = 26). SCOPE's performance was demonstrated on 211 untreated primary mesothelioma cancers and 201 treatment-resistant metastatic cancers. Finally, SCOPE was used to identify the putative site of origin in 15 cases with initial presentation as cancers with unknown primary of origin. Results: A total of 10 688 adult patient samples representing 40 untreated primary tumor types and 26 adjacent-normal tissues were used for training. Demographic data were not available for all data sets. Among the training data set, 5157 of 10 244 (50.3%) were male and the mean (SD) age was 58.9 (14.5) years. Testing was performed on 211 patients with untreated primary mesothelioma (173 [82.0%] male; mean [SD] age, 64.5 [11.3] years); 201 patients with treatment-resistant cancers (141 [70.1%] female; mean [SD] age, 55.6 [12.9] years); and 15 patients with cancers of unknown primary of origin; among the treatment-resistant cancers, 168 were metastatic, and 33 were the primary presentation. An accuracy rate of 99% was obtained for primary epithelioid mesotheliomas tested (125 of 126). The remaining 85 mesotheliomas had a mixed etiology (sarcomatoid mesotheliomas) and were correctly identified as a mixture of their primary components, with potential implications in resolving subtypes and incidences of mixed histology. SCOPE achieved an overall mean (SD) accuracy rate of 86% (11%) and F1 score of 0.79 (0.12) on the 201 treatment-resistant cancers and matched 12 of 15 of the putative diagnoses for cancers with indeterminate diagnosis from conventional pathology. Conclusions and Relevance: These results suggest that machine learning approaches incorporating multiple tumor profiles can more accurately identify the cancerous state and discriminate it from normal cells. SCOPE uses the whole transcriptomes from normal and tumor tissues, and results of this study suggest that it performs well for rare cancer types, primary cancers, treatment-resistant metastatic cancers, and cancers of unknown primary of origin. Genes most relevant in SCOPE's decision making were examined, and several are known biological markers of respective cancers. SCOPE may be applied as an orthogonal diagnostic method in cases where the site of origin of a cancer is unknown, or when standard pathology assessment is inconclusive.